Therapeutic antibodies used as biopharmaceuticals are highly complex molecules requiring strict monitoring and control during manufacturing. Due to its nature as a biological drug, drug safety and innate variation in production methods is always a cause for concern. Thus, a series of workflows for comprehensive, in-depth analytical characterization should be applied to analyze and characterize product quality.
With the approval of monoclonal antibody products dramatically increasing each year such as mAbs, Fc-fusions, Fab, antibody-drug conjugates, bispecific mAbs (bsAbs) and bispecific T cell engagers (BiTE), understanding individual post-translational modifications (PTMs) can give a better insight and prediction as to the in vivo efficacy, pharmacokinetics, and mechanism of action of manufactured Abs.
Characterization of Abs is usually performed in several perspectives, starting from top-down analysis (overall structure) to bottom-up analysis (peptide sequencing), and finally, middle-level analysis (Fc, Fv, PTMs).
To enable antibody characterization methods, ACROBiosystems has developed a series of enzymes to assist with the characterization of antibodies and their related PTMs.
|Product Details||Cat. No.||Unit Definition|
|Endo H (500U/μl)||ENH-S5116||One unit is defined as the amount of enzyme required to remove >95% of the carbohydrate from 10µg of denatured RNase B in 1 hour at 37°C in a total reaction volume of 10µl.|
|Endo S (200U/μl)||ENS-S5143||One unit deglycosylates ≥ 95% of 1 μg human IgG, when incubated in 10 mM sodium phosphate, 150 mM NaCl, pH7.4 at 37°C for 30 min.|
|SpeB (40U/μl)||SPB-S5115||One unit digests ≥ 95% of 1 µg human IgG1 when incubated in PBS ith 5 mM DTT or TCEP, pH 7.4 at 37°C for 1 hour.|
|IdeS (20U/μl)||IDS-S5143||One unit digests ≥ 95% of 1 μg human IgG when incubated in 10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, pH7.4 at 7°C for 30min.|
Tools for Glycan Analysis
The structure and type of N-linked glycans can affect the immunogenicity, pharmacokinetics, and pharmacodynamics of therapeutic proteins such as monoclonal antibodies (mAbs) and Fc fusion proteins. Enzymes such as Endo H and Endo S provide type-specific cleavages of N-linked glycans for subsequent analysis.
Endo H cleaves asparagine-linked (N-linked) hybrid or high mannose oligosaccharides but not complex oligosaccharides.
Cleavage of high mannose or hybrid N-glycans
Selective action: Does not cleave complex glycans
≥95% purity, as determined by SDS-PAGE
Reaction time: 1h
Endo S preferentially cleaves complex oligosaccharides and avoids the cleavage of asparagine-linked (N-linked) hybrid or high mannose oligosaccharides.
Cleaves complex glycans, limited activity on high-mannose and hybrid-type glycans.
Highly efficient IgG-specific endoglycosidase
≥95% purity as determined by SDS-PAGE
Reaction time: 30 min
Antibody Subunit Fragmentation
SpeB enzyme digests IgG to generate Fab and Fc fragments. Purified and homogenous Fab fragments can be used in many applications, including:
Studying the monovalent binding of Fab fragments to the antigen. Elimination of Fc-mediated effector functions. Reduced unspecific binding from Fc interactions.
Cleavage site (Human IgG1): KTHT / CPPCPAP (above the hinge)
Reaction time: 60min
IdeS protease specifically cleaves IgGs below the hinge region into F(ab´)2 and Fc fragments.
Cleavage site (Human IgG1): CPAPELLG / GPSVF (below the hinge)
Reaction time: 30 min